Gaussia luciferase kit

To determine SMAD3 activity, SW1353 cells were transfected with a (CAGA)₁₂ reporter^{33 (link)} and CMV-Gaussia as transfection control^{34 (link)} (100 ng/ul) with Fugene6 (Promega, Madison, WI, USA), according to the manufacturers’ protocol. Bioluminescence was analysed in cell lysates. Cell lysis was performed in Passive Lysis Buffer (Promega) and luciferase activity was measured on a Tristar LB 942 (Berthold) Microplate Reader using the Luciferase Assay System for Firefly luciferase ((CAGA)₁₂) (Promega) and the Gaussia Luciferase Kit (New England Biolabs).

Conditioned cell culture medium was sampled from plates (20 μl/well; MCA) at feeding and pipetted into a white 96-well plate (Greiner Bio-One) and assessed for luminescence using the stabilized Gaussia Luciferase Kit (New England Biolabs, Inc.; 50 μl of a 1:1 dilution with 18 MΩ water) and read in a plate reader (Tecan M200 PRO; 5 s 1 mm orbital mix, 45 s wait, then read 2 s/well, 20°C, no attenuation). All data were normalized against the conditioned media from pellets treated with basal chondrogenic media. Basal conditioned medium from cells was supplemented with vitamins and minerals to determine if there was any effect on the luciferase assay, none was seen (Supplementary Data S8).